Conventionally, the measurement of the amount of an analyte in a sample using a redox reaction has been utilized for a wide range of applications. For example, such measurement has been utilized for measuring glycated proteins in applications such as biochemical analyses, clinical tests, and the like.
For instance, glycated proteins in blood, particularly glycated hemoglobin in erythrocytes, are significant indicators in the diagnosis, therapy and the like of diabetes because they reflect the patient's past history of blood glucose levels. Such glycated proteins in erythrocytes are measured using the above-noted redox reaction, for example, in the following manner.
First, erythrocytes are hemolyzed to prepare a sample. This hemolyzed sample is treated with a fructosyl amino acid oxidase (hereinafter, referred to as “FAOD”) so that the FAOD is allowed to act on a glycated portion of the glycated protein, thus generating hydrogen peroxide. The amount of this hydrogen peroxide formed corresponds to the amount of the glycated protein. Then, a peroxidase (hereinafter, referred to as “POD”) and a substrate that develops color by oxidation (a chromogenic substrate) further are added to this sample, so that a redox reaction occurs between the hydrogen peroxide and the chromogenic substrate with the POD as a catalyst. At this time, since the oxidizing substrate develops color when it is oxidized, the amount of the hydrogen peroxide can be determined by measuring the color developed. As a result, the amount of the glycated protein in erythrocytes can be determined.
However, various kinds of reducing substances such as ascorbic acid (AsA) and bilirubin usually are present in blood. Moreover, various kinds of reducing substances such as glutathione (GSH) are present in erythrocytes. These reducing substances may, for example, reduce the hydrogen peroxide, may inhibit the redox reaction, or may reduce the reducing agent after it develops color, which causes degradation of the color. Therefore, there has been a problem that it is difficult to determine the amount of the glycated protein in erythrocytes accurately.
Also, there has been another problem that the accuracy of the determination may deteriorate because the concentration of the contained reducing substances is not constant in each sample.
In order to avoid these problems, for example, various kinds of oxidizing agents have been added to samples. For example, JP 56(1981)-151358 A discloses a method of using halogen oxides such as iodic acid or periodic acid as oxidizing agents. JP 57(1982)-13357 A, JP 57(1982)-161650 A, JP 59(1984)-193354 A, JP 62(1987)-169053 A and JP 3(1991)-30697 A also disclose methods of using complexes of metals such as cobalt, iron, cerium, etc. as oxidizing agents.